Sulfonamide and nitroimidazole compositions for control of pasteurella infections in warm blooded animals and methods of use

ABSTRACT

A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 2AMINO-5-(5 - NITRO-1-SUBSTITUTED-2-IMIDAZOLYL)-1,3,4-THIADIAZOLES AND OXADIAZOLES WHEN COMBINED WITH A SULFONAMIDE RESULTS IN A COMPOSITION DEMONSTRATING A SYNERGISTIC EFFECT IN THE CONTROL OF PASTEURELLA INFECTIONS IN WARM-BLOODED ANIMALS.

U.s. cram- 229 POSITIONS FOR CONTROL OF PASTEURELLA INFECTIONS IN WARM BLOODED ANIMALS AND METHODS OF USE Richard Jay Shirk, Trenton, and Gordon Arthur- Kemp,

United States Pate 7 follow a chronic 'course or may be an acute septicemia.

Princeton, N.J.-,-'assignors to American'Cyanamid Com pany, Stamford, Conn. No Drawing. Filed Mar. 8, 1971, Ser. No. 122,199 Int. Cl. A61k 27/00 ABSTRACT OF THE DISCLOSURE A compound selected from the group consisting of 2- amino-S-(S nitro-1-substituted-2-imidazolyl)-1,3,4thiadiazoles an d oxadiazoles when;combined with a sulfona 7 Claims amide results in a composition demonstrating a synergistic effect in the control of Pasteurella infections warm-blooded animals.

BRIEF SUMMARY OF THE INVENTION The present invention relates to novel synergistic com positions, effective forvcontrolling .F'asteurella infections warm-blooded animals, containing (1 a compound DETAILED DESCRIPTIONZOF THE lNYENTION Although the pathogenicity of Pasteurella organisms for warm-blooded animals has been recognized for about.

seven decades there is still no entirely satisfactory method ed' from the group'consisting of Z-arnino-S-(i-Ilitro- ,1 substituted kimidazolyl)fl;3 4 thiadiazoles and oxadiav.Z Qles an d, 2.) a sulfonamide; The invention; also relates teurella infections in warm-blooded animals.

for controlling Pasteurella infections in-such animals.

Moreover, there is no entirely satisfactory method. of immunizing warm-blooded animals against-attack from these microorganisms.

As early as 1880, Louis Pasteur developed'a' vaccine which was partially successful in protecting chickens againstPqsteigrella mult cida thecausatiye agent forfowl cholera. However, protection was short lived and other 'methods of control have been sought. In recent years, sulfa drugs and a nitrofuram'have been used to combat fowlcholeraand' other Pasteurella infections but these treatments have had only v ery limited success. If, therefore, a method could be provided whereby eifective convtrol of Pasteurella infections was obtained, such method Susceptible to the organism are turkeys, rams, silver foxes, swine, mice, wild rats, dogs, cats, and horses.

Acute Pasteurella multocida infections are generally characterized by a septicemia accompanied by bloodvascular congestion, enteritis and submucous and subserous hemorrhage.

The organism is the causative agent for fowl cholera and is frequently responsible for serious losses in flocks of chickens, ducks, geese and turkeys. Outbreaks of the disease have likewise been reported in flocks of wild ducks and pheasants.

This organism is also known to produce hemorrhagic septicemia typified by pleuropneumonia in cattle, sheep, swine, rabbits, mink and other warm-blooded animals.

Pasteurella hemolytica, another organism of the Pasteurella family, produces a difluse pneumonia in sheep and cattle and may be marked by a fibrinous pleurisy with adhesions.

Pasteurellal pestis has been associated with the bubonic ,plague and is pathogenic to man and many species of rodents including the rat, squirrel, rabbit and wild guinea pig. Pasteurella pestis infections have also been reported in;cats,dogs-horses, cattle,-sheep, goats, and wild birds.

Pasreurgllae:tularensis has been *fOUndftO produce natural infection in wild rabbits and haresthroughout the 'United. States;.ground squirrels in California and Utah;

.beaver in rM'ontana; chipmunk-and sheep" in Idaho; quail and grouse-in Minnesota; wild rabbit's-in Iapan,'Canada,

"carnivora" that have eaten infected rabbits. Tularemia is a fatal septicemia in rodents. White mice, rabbits, cotton rats, hamsters, and guinea pigs are all susceptible.

Surprisingly, we have now discovered a method of and synergistic composition for the effective control of Pas- In its broadest aspects, the present invention involves the treatment of warm-blooded animals with a therapeutically effective amount of a composition containing (1) a compound having the formula:

wherein X'issulfur or oxygen; R is a member selected from the group consisting of loweralkyl C -C and preferably methyl or ethyl, hydroxy loweralkyl such as hydroxymethyl, hydroxyethyl or hydroxypropyl and R and R each represent hydrogen or lower alkanoyl; and

e giroup 'sulfaguanidine, sulfadiazine, sulfanilamide, sulfathiazole, sulfa;

quinoxaline and 3 sulfanilamido-6-ethoxypyridizine.

Advantageously, the compositions of the present invention can be administered to warm-blooded animals admixed with their feed or as a top dressing for the feed. They may also be made up in tablets, capsules, boluses, pills and the like or as drenches or liquid suspensions and orally administered to the animals on an individual basis. If desired, the synergistic compositions of the present invention may also be prepared as injectable solutions and administered parenterally to the animal host.

When administered in the animal feed, we have found that generally from about 6.25 to 100 p.p.m. and preferably 12.5 to 50 p.p.m. of the nitroimidazolyl thiadiazole or .oxadiazole and fromabout 31.5 to--1000 p.p.m. and preferably 125 .to 550 p.p.m. of the sulfonamide, gives excellent control of Pasteurella infections.- If ad-" ministered as a single oraldose, generally from":2-.5'to 40 mg./kg. of-body weight and preferably S'to 20 rug/kg. of the nitroimidazoyl'thiadiazole or oxadiazole and from 40 to 80 mg./kg. of body weight of the sulfonamide is effective for controlling Pasteurella' infections insaid'an'i mals. Similar dosages are also effective when the'comp'o' sitions are administered parenterally to the animal hosts.

' DESCRIPTION OF THE PREFERRED EMBODIMENTS In all of the following examples, the poultrydiet set TAM M ID R -MIX FOR. DI T .""TE. T. ".T -'---:T--::--.-1--:m r 125 dl-Methionine g 500 Vitamin A U 3300 Vitamin D I.C.U 1100 Vitamin E T U 2.2 Vitamin B g 11.0 Riboflavin mg 4.4 Niacin mg 27.5 Panthothenic acid mg 8.8 Choline chloride ....mg 500.0 Folic acid m 1.43 Menadione Na. bisulfate mg 1.10

Ground yellow corn to 5 gms.

Example 1 Six-dayfildchicks were removed from th e 95 holdin'g'roorn to the .test room tmaintained at180- 82 F.)

- and distributed into uniformlweight; groups of: 10 to 20 birds each.. -As each group was assembled it was'placed' I The following day the birds were treated and infected;

forth below was employed unless it is expressly stated'to the contrary.

they were continued on the untreated diet throughout the remainder of the test, which was 12 days postinfection. Birds weretrea ted with the indicated drug by 'gavage as a single doseof 0.5 ml. immediately prior" to infection. The indicated drug levels were prepared by suspending or dissolving the drug in sterile distilled water containing 0.1% agar.

TABLE L-EXPERIMENTAIJ PASTEURELLA'MULTOCIDA-INFECTION IN WEEK-OLD CHICKS .l- TITRh'lflQN DATA. V

Survivors/total chicks on test-12 days postlntectlon i Test numbei i Summary Infection route Dilution 1 2 4 -5 a 12 13 14 15 16 17, s T cents Air Sac ioo zo v4 20 0 20 a 10+ 0 20 2 20 1 20 4 I0 o 2o 2/20 rm 4 10*" one 7 20 2 20 13 0 Infection: The sev en-dayl ol d chicks wereinfected by POULTRY DIET Ground yellow corn Soybean oil meal protein) Menhaden fish meal protein) Trace minerals 2% zinc Vitamin/amino acid pre-mix multocida culture diluted 10- in'trypticase soy br h' injecting into' the left air-sac 0.2 ml. of a five houfr Plate counts obtained by counting ,colonys on tryptiicase soy agar indicated that'in'this "series of 15 tests the average infective dose was 11x10? (range 5 to 2 5 '1'0 colony formingfunits /chick. Data obtained are reported 'i-b'elow.

when injected via air sac into chicks.

The data-of Table II provide 'eflicacy data obtained with graded levels of test drugs administered alone and establish basis for comparison with the drug combina tions of the present invention which are-presented in the ensuingexamples. w p Y SIT Test number.

Peree Treatment:

A=2-aruir1o-5- (l-meth yl--nltr0- 2-imidazolyD-1,3,4-tl1ia.diazole: Mgdkg-S O D 2.5 B Chlortetracycline hydrochloride: Mglkg.-SOD:

S/I=Survivors/Total birds tested. "MST=Mean Survival Time (days) of birds that died.

TABLE IV.-EVALUATION OF DRUGS IN COMBINATION AGAINST A PASTEURELLA MULTOC'IDA INFECTION IN WEEK-OLD CHICKS Total" S,-total f MST" and distributed into uniform weight groups of 10 to 20 birds each. As each group was assembled it was placed in an individual cage of a battery consisting of: five such cages in tiers. After the birds were grouped and assigned to cages they were given water but no diet for the next Test number P ent 26 hours Q ere 4 Treatment: 19 20 Total 5 Three hours before the infections, the starved birds m were given diets containing appropriate levels of testdrugs. 5- 11Z 5 9 y After three hours feeding, the bll'dS were 1nfected and conii i a gf' l V 3 V tinued on the drug diets for. five days at which time they 10 33 1 Q2 again received .the'unmedicated control diet. 28 Infections were obtained by injecting into the left air g sac of birds 0.2 ml. of a five-hour P. m'ultocida culture dig f fig g fi .luted 10- in trypticase soy broth as described in Example Mg'IkgPSOD: 11 20 11 20 22 40 3 0 above l I I: I :1: 1 20 3 m 4 40 10 1 Inthis fieries oftests to evaluate combinations of Com- C p 1 s 9/19 2M0 39/39 0 pound A and sulfonamides, favorable mteractions were 95 "51', demonstrated ja's indicated in the tables. In this series, 3g g-g Compound A had a median effective dose of 27 p.p.m. in

I the diet, sulfamethazine (C) 4 89; p.p.m and sulfaethoxy- 8g 50 pyridazine (D) 249 p.p.m. A diet level0f'12.5 p.p.m. of 73 5 (A) was ineffective by itself but in combination with g graded levels of the sulfonamides significantly reduced the 100 median effective dose of both sulfamethazine and sulfa- S/T =Survivors/Total birds on test 12 days postiniection. "MS'I=Mean Survival Time (days) of birds that died;

'N0rE.-Average infective dose/bird was 13 10 organisms.

F Eirample 3 TABLE V.EXPERIMENTAL PASTEURELLA MULTOO'IDA 1101 1000 1010 IN WEEK-om) clitoris-EFFECT 0F DRUo COMBINATIONS Average feed con- Average drug sumption, consumption, Percent Number g./kg./3 hrs. mg./kg./3 hrs. Survivors sur- Median effective dose p.p.m. in diet ED Diet treatment (p.p.m.) of tests .3 to 0 hr. .3 to 0 hr. per total vivors confidence limits) Compound A: l I

TABLE V-Continued Average feed con- Average drug sumption, consumption, Percent Number g./kg./3 hrs. mg.lkg./3 hrs. Survivors sur- Median efieetive dose p.p.m. in diet EDro Diet treatment (p.p.m.) of tests .3 to hr. .3 to 0 hr. per total vivors (95% confidence limits) 2 105 53+2. 6 93 112 (001-207) plus 25 p.p.m.

2 104 52+1.3 35/40 88 273 (205-357) plus 12.5 p.p.m.

2 108 54+0.7 29/40 73 421 (245-750) plus 6.25 p.p.m.

Compound A CH; 1,3,4-thladiazole, 2-am1no-5-(l-methy1-5-n1tro-2-1mldazo1y1) N S NOW h TNH: L h N Compound B HC1= Chlortetracyclinehydrochloride.

01 OH: OH N(OH:)1

OH OH C ONE:

N NHg-Q- s Or-NH Compound D 3-su1lan1lam1de-0-ethoxypyrldaz1ne.

NH -8 O3NH OCQH;

N Sulfaullamlde, N'-(6-ethoxy-4-pyr1m1d1nyl). NHa-Q- s 04-NH-QN 2 TABLE VL-EXPERIMENTAL PASTEURELLA MULTOC'IDA INFESTION IN WEEK-OLD CHICKS-EFFECT OF DRUG Compound 0 2-su1fanllam1do-4,6-d1methy1pyr1mld1ne=Sulmmethazme.

Compound E COMBINATION Average feed con- Average drug sumption, consumption, Percent Number g./kg.l3 hrs. mg.lkg./3 hrs. Survivors sur- Median efl'ective dose, p.p.m. in diet EDm Diet treatment (p.p.m.) of tests .3 to 0 hr. .3 to 0 hr. per total vivors (95% confidence limits) Compound A:

2 114 114 2.9 39/40 98 67 (24-103) plus p.p.m. A.

2 120 120+1. 5 /40 100 131 (98-164) plus 12.5 p.p.m. A.

2 117 58. 5+0. 7 38/40 176 (96-319 plus 6.25 p.p.m. to 5.55.

Infected-untreated 9 115 3/180 2 Normal 9 115 179/179 TABLEVII.EXPERIMENTAL PASTEURELLA M ULTOC'IDA INFECTION IN WEEK-OLD CIIIOKS EFFECT OF DRUG C OMLBIVNVATIONS.

, .1 wvt -11 .1- H .1

feed consumption, Number g./kg./3 hrs. Median effective dose Diet treatment (p.p.m.) of tests .3 to hr confidence limitsj 0 (1A: 1- 1 M11. a v oniggun 1 104 100 27 (24-30).

2 111 so 2 110 53 2 109 as ,2 103 a 1 101 25.3+5.1 1 102 12.8+5.1 A V I M 1 s "f '100" '6.'3+5.0 1 94 3.0+4.s 00 1 108 27. 0+2.7 23 (limits not calculable) plus p.p.m. A. 1 110 13.s+2.s 5 1 105 6.6+2.0 1 94 2.9+2.4 13/20 65 1 94 23. 5+L2 20/20 100 60 (limits not calculable) plus 12.5 p.p.m. A; 1 106 1a.a+1.3 13/20 ""55 1 10s 0. 8+1. 4 10/20 50 1 107 3.3+L3

2 115 23.0 39/40 0 9s e0, 40-9s). 2 108 10.8 /40 as 1 1 .s 2 .9 1 .7

Infected-untreated. 9 Norm 9 Example 4 k Example 5 The surprising enhancement in the effectiveness of the .Pfollowing the procedure of Ekample 1 above, the comcombination of nitroimidazolyl thiadiazoles or oxadiazoles 35]; pound 2-(5-amino-1,3,4-thiadiazol 2 yl)-5-nitro-1-imidwith sulfonamides, over the individual components, for agolethanol, identifiedasCompound F, is used alone and the control of fowl cholera in poultry is demonstrated by, in combination; with 2-siil fanilamido-4;6 diniethylpyrinii the following tests. 5 titling" .e. Compouhd.:..C,-/against Pasteurella infection in In these tests, weak-old chicks are divided into groups chicks. The data obtained are reported in Table IX below of 20, weighed as groups and placed in pens. Infections and demonstrate therenhanced efiect-obtained with the and treatments are made as described in Example 1, ex combination k; cept that a variety of drugs are used alone and in com- 7 TABLE IX bination with sulfonamides, to determine the efiicacy 0f Pasteurella multocida RC 315 infection in week-old chicks efiectof com id drugs d d 1 bi i af h controlling 1 a Jainations--=-oi- =sullametl1azlne a11d-- -2-(S-amlno-l,3,4-thladiazobZ-yl) 5-nltrol-lmidazolethanol cholera.

Data obtained are given in the tables below. These SIT Percent data demonstrate:

"1' a x p (1) Ihat manyantihacterialswhich are highlyefiective 234331 8 agalnst a wide variety of bacterial infections in animals are not effective against Pasteurella infections in poultry, Q 9/20 45 1 1. 4/20 20 even when admlnistered at relat1vely h gh levels, (2) These inefifectiveflantibacterialspare not made efli-p 120995 9??? F= 10 mgJkg 20/20 100 cacious by the use of sulfonamldes m combmatlon there- 5I11g-lk 20/20 100 with and, Y l 2.5mg.kg 15 20 75 I 1 1.25 9 2 (3) That the efiicacy of 2-am1no-5-(l-methylJ-mtro-v C i f 0 45 Z-imidazolyD-1,3,4-thiadiazole is unexpectedlyenhanced mp9un 1 Y I c undF 10 .1; 1 by the use thereof 1n combination with a sulfonamide. 0833811511 my 183 Compound 85 TABLE VIII Compound 90 survi OompoiindC 4 Single oral 25: s'fi- Compound ,lflmgJkg---" 20/20 100 Compound dose, mgJkg. total vivbrs Compound 5 g-lk 20/20 1 Compound F, 2.5 mgJk 16/20 80 Uninfected-lmtreated 60/60 100., C mp d F, 1. 5 mgJkgmn 8/ 0 v 40 2 60, 1 5 x i n V l 1 40 1; g 1 Significant at p==i05 over drug levels V j I 40 V 0 NOTE-Conclusions: Indicates enhanced efiect the indicated levels. 40 0 20 0 40 0/20 0 I We cla11n: w o 8 gg 2 1. A l synergistic composition for the control of Pasteugggpggg g 1 3? 21 /38 22 rella infections in warm-blooded animals comprising, (a) P f from 2l'5 to 40 mg./kg. of animal body weight of a com CompoundCplusv Tylosm SOHO 7/207 :35 Ound seIected from the group having the formula Penicillin (3-. 804-40 8 20 '40 n Neomycin s0+40 3 20 40 R,

Streptomycin +40 9/20 45 N w "'X 7 Furazolldone- 80-1-80 9/20 :45 OzN N Do 80+160 4 20 20,, q 1

CompoundA 80+1.25 20/20 R} wherein X is sulfur or oxygen, R represents a member selected from the group consisting of lower alkyl C -C and hydroxy lower alkyl having 1 to 4 carbon atoms and R and R each represent hydrogen or lower alkanoyl and (b) from 40 to 80 mg./kg. of animal body weight of a sulfonamide selected from the group consisting of sulfamethazine, sulfamerazine, sulfadiazine, sulfathiazole, sulfaquinoxaline, 3-sulfanilamido-fi-ethoxypyridazine, N- (6-ethoxy-4-pyrimidinyl -sulfanilamide.

2. A composition according to claim 1 where, in the imidazolyl formula, R is methyl or hydroxyethyl, R is hydrogen and R is hydrogen or COCH 3. A medicated animal feed composition efiective for controlling Pasteurella infections in animals ingesting said feed, comprising, an edible feedstutf, from 6.25 to 100 p.p.m. of a compound of the formula:

wherein X is sulfur or oxygen, R is a member selected from the group consisting of loweralkyl C -C and hydroxy lower alkyl C -C and R and R each represent hydrogen or lower alkanoyl and from about 31.5 to 1000 p.p.m. of a sulfonamide selected from the group consisting of sulfamethazine, sulfamerazine, sulfadiazine, sulfathiazole, sulfaquinoxaline, 3-sulfanilamido-6-ethoxypyridazine, N'-(6-ethoxy-4-pyrimidinyl)-sulfanilamide.

4. A medicated feed composition according to claim 3 where, in the imidazolyl formula, R is methyl or hydroxyethyl, R is hydrogen and R is hydrogen or CO-CH 5. A method for controlling Pasteurella infections in warm-blooded animals comprising, administering to a host animal a composition containing (a) from 2.5 to 40 mg./

14 kg. of animal body weight of a compound having the formula:

{here X is sulfur or oxygen; R is loweralkyl C -C and 1 and R, are each selected from the group consisting of hydrogen and loweralkanoyl and (b) from to 80 mg./kg. of animal body weight of a sulfonamide selected from the group consisting of sulfamethazine, sulfamerazine, sulfadiazine, 3-sulfanilamide-G-ethoxypyridazjne, N' (6-ethoxy-4-pyrimidinyl)-sulfanilamide.

6. A method according to claim 5 wherein the imidazolyl is 2-amino-5-(l methyl-S-nitro-Z-imidazolyl)-1,3,4-- thiadiazole, the sulfonamide is sulfametha zine and the composition is administered as a single oral dose providing from 5 to 2 0 mg./kg. of body weight of the thiadiazole and from 40 to 80 mg./kg. of body weight of sulfamethazine.

7. A method according to claim 5 wherein the Pasteurella organism is Pasteurella multocida and the host animal is a fowl. v p

References Cited Redin et aL: (988381)); Burden et al.: (98839q); Carvajal et al.: (98840h); Burden et al.: (98841i); Chem. Abst., vol. 72 (1970) (numbers in parentheses are abstract numbers).

Bachmann et al.: Chem. Abst., vol. (1970), abst. No. 990752.

SAM ROSEN, Primary Examiner US. '01. X.R. 424-470 

